1,3-丙二醇发酵过程中乳酸抑制的研究

目的:研究乳酸对克雷伯氏肺炎杆菌(Klebsiella pneumonia)产1,3-丙二醇的影响。方法:通过在摇瓶和反应器水平下分析不同菌株(包含无乳酸、2,3-丁二醇产生的基因敲除菌)的乳酸代谢特性。结果:前期添加6 g/L的乳酸使1,3-丙二醇的产量降低了19%,而发酵10h后添加乳酸几乎不表现出抑制作用。通过对乳酸敲除菌株的代谢分析发现,发酵后期能够消耗培养基中的乳酸,这在一定程度上也反映了菌体发酵后期对乳酸的耐受性。结论:乳酸的抑制作用主要发生在1,3-丙二醇发酵的前期。解除了一株无副产物2,3-丁二醇生产株前期乳酸的过早积累后,1,3-丙二醇的的产量提高了56%。     

D-乳酸合成阻断对肺炎克雷伯氏菌的1,3-丙二醇发酵的影响

利用Red同源重组技术,快速敲除肺炎克雷伯氏菌中的编码D-乳酸脱氢酶的两个基因——ldhA和dld,获得KG1-1和KG1-2两个突变株,并研究了敲除编码D-乳酸脱氢酶基因丧失合成D-乳酸的KG1-1菌株的1,3-PD产量和菌体生长变化,实验结果表明乳酸合成缺失对现有工艺1,3-丙二醇发酵无影响。     

利用PDOR同工酶基因yqhD对产1,3-丙二醇克雷伯氏杆菌进行基因工程改造

由于Klebsiella pneumoniae 1,3-丙二醇合成途径中,加强甘油脱水酶基因表达,导致因NADH供应不足使3-羟基丙醛累积,并对菌体生长及1,3-丙二醇合成造成负面影响。为改善Klebsiella pneumoniae 1,3-丙二醇合成途径,本文利用PCR技术从大肠杆菌(Escherichia coli)中扩增出以NADPH 为辅酶的1,3-丙二醇氧化还原酶同工酶编码基因yqhD,从克雷伯氏杆菌中扩增出2.66kb的甘油脱水酶基因（dhaB）,构建了产1,3-丙二醇关键酶基因的串联载体pEtac-dhaB-tac-yqhD,并将其转入到野生克雷伯氏杆菌(Klebsiella pneumoniae)中,重组载体得到了表达。通过初步发酵,重组后的克雷伯氏杆菌产量比原始菌高20%左右,副产物中乙酸和丁二醇分别下降30%左右。     

增强胞内NDAH水平和乙偶姻还原酶活力提高2,3-丁二醇产量

枯草芽孢杆菌Bacillus subtilis 168是一株安全生产菌株,首次通过弱化B.subtilis 168磷酸戊糖途径(PPP)中的关键酶葡萄糖-6-磷酸脱氢酶(G6PDH)基因zwf,研究了其对胞内NADH水平的影响,进而研究其对2,3-丁二醇(2,3-BD)及副产物合成的影响。弱化菌株B. subtilis168△zwf进行摇瓶发酵实验,与出发菌株相比,胞内辅酶NADH水平得到了增强, 2,3-BD产量提高了15.0%,主要副产物AC积累量下降了10.6%,但乙酸、乳酸等有机酸的积累量提高。为了进一步提高2,3-BD生产效率,在B. subtilis168中克隆表达了不同来源的ACR基因,研究发现克雷伯氏菌来源的ACR酶活力最高,将此来源的ACR的基因kphs克隆到B.subtilis168△zwf中加强表达,对重组菌株B.subtilis168△zwf/pMA5-kphs进行摇瓶发酵实验,与出发菌相比,2,3-BD产量提高了37.3 %,主要副产物AC积累量下降了28.1%,同时,乙酸等分支路径的其他副产物也有不同程度的降低。     

产2,3-丁二醇高木糖耐性肺炎克雷伯氏菌转录组学分析与发酵优化

选育高木糖耐性肺炎克雷伯氏菌并对其进行转录组学分析与产2,3-丁二醇发酵条件的优化。首先通过适应性进化定向筛选出了具有高木糖耐受能力和2,3-丁二醇产量提高的肺炎克雷伯氏菌(KP2),然后对其进行转录组学分析与发酵条件优化。结果显示,筛选出的高木糖耐性的肺炎克雷伯氏菌木糖的转化率提高了174.5%,2,3-丁二醇的产量提高了227%。转录组分析结果表明亲本菌株KPG和KP2菌株差异表达基因中有242个基因上调和263个基因下调。Pathway分析表明,转录水平的变化主要表现在信号转导、碳水化合物代谢、能量代谢和其它物质代谢等。在1L反应器中,初始木糖浓度120 g/L时最优发酵条件为:温度35℃,转速230 r/min,通气量0.7 L/min,最适pH值5.9。在最优条件下,2,3-丁二醇产量达43.75 g/L,相比优化前提高了32.7%。本研究获得了高效利用木糖生产2,3-丁二醇的菌株,对表型变化产生的机理在分子水平上进行阐述,并得到了最优的发酵条件,结果可以为2,3-丁二醇的生物转化提供参考,对2,3-丁二醇的生物法生产具有一定的指导意义。     

聚羟基丁酸路径在克雷伯氏菌中的构建

以生物柴油的副产物甘油生产高附加值的1,3-丙二醇,现已成为提升生物柴油产业链经济性的重要途径,而中间代谢产物3-羟基丙醛积累造成细胞死亡,发酵异常终止是生物法生产1,3-丙二醇过程中的关键问题。不同于传统的降低3-羟基丙醛积累的思路,本文从增强克雷伯氏菌对3-羟基丙醛的抗逆性出发,改善克雷伯氏菌1,3-丙二醇的生产性能,首次将聚羟基丁酸路径引入克雷伯氏菌中,构建了新型基因工程菌,并对其1,3-丙二醇发酵性能及聚羟基丁酸代谢进行了初步的研究。经IPTG诱导,工程菌中检测到聚羟基丁酸,其含量随IPTG浓度增加而增大。优化的IPTG浓度为0.5 mmol/L。初始甘油50 g/L时,野生菌可正常发酵生产1,3-丙二醇,1,3-丙二醇浓度达到22.1 g/L,其质量得率为46.4%。当初始甘油达到70 g/L时,由于高浓度3-HPA积累,野生菌发酵终止,而工程菌可正常发酵生产1,3-丙二醇,PDO产量可达31.3 g/L,其质量得率为43.9%。同时检测到聚羟基丁酸积累。研究结果有助于加深对克雷伯氏菌1,3-丙二醇代谢机理的认识,为克雷伯氏菌的进一步优化提供了新的思路。     

异源表达木糖异构酶基因对克雷伯氏菌合成1,3-丙二醇的影响

【目的】提高克雷伯氏菌胞内还原力以强化1,3-丙二醇合成。【方法】将来源于大肠杆菌的木糖异构酶基因在克雷伯氏菌中异源表达,构建重组菌。研究重组菌添加不同浓度木糖为辅底物与甘油共发酵过程中代谢产物和NADH的变化规律。【结果】与对照菌相比,重组菌细胞内还原力NADH提高了0.1?0.3倍,1,3-丙二醇产量达到23.31 g/L,提高20%,1,3-丙二醇转化率从0.60 mol/mol提高到0.73 mol/mol。【结论】木糖异构酶基因的表达强化了木糖代谢途径,经磷酸戊糖途径积累大量还原力,促进了1,3-丙二醇的生成。     

克雷伯氏菌产1,3-丙二醇ldhA基因缺失菌株的构建

克雷伯氏菌(Klebsiella pneumonia)甘油歧化发酵生产1,3-丙二醇(1,3-PD)的过程中,乳酸是氧化途径最主要的副产物,乳酸的产生和积累,不仅限制了菌体本身的生长,而且严重影响了1,3-丙二醇的转化率。利用λRed重组技术对Klebsiella pneumonia中的酶乳酸脱氢酶基因(ldhA)进行改造。在λRed重组系统作用下,将带有300 bp的线性同源片段ldhA1-Cm-ldh A2与基因组DNA的同源重组,经过抗性筛选和PCR鉴定最终获得了ldhA基因缺失菌株K.pneumonia2-1ΔldhA。经过24 h发酵可知,乳酸最大产出浓度由原来的10.16 g/L降为0.49 g/L,1,3-PD由原来的78.83 g/L增长为85.76 g/L,甘油转化率由60.64%增长到65.97%,提高了5.33%。     

产1,3-丙二醇菌株的诱变和筛选

为提高克雷伯氏肺炎杆菌产1,3-丙二醇的能力,以离子束、紫外线和氯化锂为复合诱变法,建立了产酸圈和产物耐受相结合的平板筛选方法,获得可耐受高浓度1,3-丙二醇并且副产物中乙醇含量较少的优良突变菌株2株。与出发菌株相比,两株高产突变菌株Klebsiella pneumoniae LM 03和Klebsiella pneumoniae LM05的1,3-丙二醇产量分别提高了33% 和30% ,达到66.74 g/L和65.12 g/L;乙醇产量分别降低了38% 和24% ,降低为6.59 g/L和8.05 g/L。同时测定了诱变前后还原途径中甘油脱水酶（GDHt）和1,3-丙二醇氧化还原酶（PDOR）的酶活变化,研究表明诱变对GDHt有明显的促进作用,而对PDOR的影响不明显。该诱变和筛选方法目标明确、易操作、效率高,在1,3-PD工业规模的生物法生产中将具有良好的应用价值,而且对于其他具有工业应用价值的菌株筛选工作也具有一定的借鉴意义。     

地衣芽孢杆菌高产2,3-丁二醇菌株的选育和发酵研究

目前2,3-丁二醇生产菌株大部分为致病菌,对人类健康和环境具有一定威胁。从牛奶样品中分离到1株产2,3-丁二醇的芽孢杆菌127-7,分析其16S rRNA基因序列,确定该菌株为地衣芽孢杆菌(Bacillus licheniformis)。进一步对菌株127-7进行紫外诱变,筛选耐受高浓度葡萄糖和高产乙偶姻的菌株。摇瓶发酵结果显示,突变株BL41的2,3-丁二醇产量较出发菌株127-7提高了41.1%。对发酵副产物分析发现,不控制发酵液pH可以显著降低乳酸产量,2,3-丁二醇产量在72 h达到81.4 g/L。进一步调整补糖策略,维持最低残糖浓度为30 g/L,菌株BL41产2,3-丁二醇83.4 g/L,最高产率为1.9 g/L·h,发酵时间缩短至46 h。结果表明,地衣芽胞杆菌BL41可以作为候选菌株,用于工业规模2,3-丁二醇的生产。     

Enhanced 2,3-butanediol production in recombinant Klebsiella pneumoniae via overexpression of synthesis-related genes

2,3-Butanediol (2,3-BD) is a major metabolite produced by Klebsiella pneumoniae KCTC2242, which is a important chemical with wide applications. Three genes important for 2,3-BD biosynthesis acetolactate decarboxylase (budA), acetolactate synthase (budB), and alcohol dehydrogenase (budC) were identified in K. pneumoniae genomic DNA. With the goal of enhancing 2,3-BD production, these genes were cloned into pUC18K expression vectors containing the lacZ promoter and the kanamycin resistance gene to generate plasmids pSB1-7. The plasmids were then introduced into K. pneumoniae using electroporation. All strains were incubated in flask experiments and 2,3-BD production was increased by 60% in recombinant bacteria harboring pSB04 (budA and budB genes), compared with the parental strain K. pneumoniae KCTC2242. The maximum 2,3-BD production level achieved through fedbatch fermentation with K. pneumoniae SGJSB04 was 101.53 g/l over 40 h with a productivity of 2.54 g/l.h. These results suggest that overexpression of 2,3-BD synthesisrelated genes can enhance 2,3-BD production in K. pneumoniae by fermentation.     

Improved 1,3-propanediol production by engineering the 2,3-butanediol and formic acid pathways in integrative recombinant Klebsiella pneumoniae

In the biotechnological process, insufficient cofactor NADH and multiple by-products restrain the final titer of 1,3-propanediol (1,3-PD). In this study, 1,3-PD production was improved by engineering the 2,3-butanediol (2,3-BD) and formic acid pathways in integrative recombinant Klebsiella pneumoniae. The formation of 2,3-BD is catalysed by acetoin reductase (AR). An inactivation mutation of the AR in K. pneumoniae CF was generated by insertion of a formate dehydrogenase gene. Inactivation of AR and expression of formate dehydrogenase reduced 2,3-BD formation and improved 1,3-PD production. Fermentation results revealed that intracellular metabolic flux was redistributed pronouncedly. The yield of 1,3-PD reached 0.74 mol/mol glycerol in flask fermentation, which is higher than the theoretical yield. In 5 L fed-batch fermentation, the final titer and 1,3-PD yield of the K. pneumoniae CF strain reached 72.2 g/L and 0.569 mol/mol, respectively, which were 15.9% and 21.7% higher than those of the wild-type strain. The titers of 2,3-BD and formic acid decreased by 52.2% and 73.4%, respectively. By decreasing the concentration of all nonvolatile by-products and by increasing the availability of NADH, this study demonstrates an important strategy in the metabolic engineering of 1,3-PD production by integrative recombinant hosts.     

Improved Production of 2,3-Butanediol in Bacillus amyloliquefaciens by Over-Expression of Glyceraldehyde-3-Phosphate Dehydrogenase and 2,3-butanediol Dehydrogenase

Background

Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production.

Methodology/Principal Findings

In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD⁺ to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD⁺. In this study, to improve 2,3-BD production, we first over-produced NAD⁺-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h.

Conclusions/Significance

Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.     

生物法生产2,3-丁二醇研究进展

2,3-丁二醇是一种重要的化工原料,可广泛应用于多个领域。二战期间由于合成橡胶需要大量1,3-丁二烯,2,3-丁二醇生产空前发展。近年来,由于聚对苯二甲酸丁烯树脂、γ-丁内酯,Spandex弹性纤维及其前体的需求增长,2,3-丁二醇的需求和产量也稳步增长。多年来,生物法生产2,3-丁二醇虽然得到了广泛的研究,但一直没有实现工业化。本文从产生2,3-丁二醇的菌种及2,3-丁二醇的生理意义、代谢途径、旋光异构体的形成机理、影响发酵的因素与产物的提纯等方面对生物法生产2,3-丁二醇进行了综述并提出了生物法生产2,3-丁二醇要解决的几个问题。     

The effects of cell recycling on the production of 1,3-propanediol by Klebsiella pneumoniae

The effects of both biomass age and cell recycling on the 1,3-propanediol (1,3-PDO) production by Klebsiella pneumoniae were investigated in a membrane-supported bioreactor using hollow-fiber ultrafiltration membrane module in two separate experiments. It was determined that older cells have a negative effect on 1,3-PDO production. The concentrations of by-products, such as acetic acid and ethanol, increased in cultures with older cells, whereas the concentrations of succinic acid, lactic acid and 2,3-butanediol decreased. The effect of cell recycling was comparatively studied at a cell recycling ratio of 100 %. The results showed that cell recycling had also negative effects on 1,3-PDO fermentation. It was hypothesized that both cell recycling and biomass age caused metabolic shifts to undesired by-products which then inhibited the 1,3-PDO production. On the other hand, the use of hollow-fiber ultrafiltration membrane module was found to be very effective in terms of removal of cells from the fermentation broth.     

Fermentation and evaluation of Klebsiella pneumoniae and K. oxytoca on the production of 2,3-butanediol

Klebsiella is one of the genera that has shown unbeatable production performance of 2,3-butanediol (2,3-BD), when compared to other microorganisms. In this study, two Klebsiella strains, K. pneumoniae (DSM 2026) and K. oxytoca (ATCC 43863), were selected and evaluated for 2,3-BD production by batch and fed-batch fermentations using glucose as a carbon source. Those strains' morphologies, particularly their capsular structures, were analyzed by scanning electron microscopy (SEM). The maximum titers of 2,3-BD by K. pneumoniae and K. oxytoca during 10 h batch fermentation were 17.6 and 10.9 g L(-1), respectively; in fed-batch cultivation, the strains showed the maximum titers of 50.9 and 34.1 g L(-1), respectively. Although K. pneumoniae showed higher productivity, SEM showed that it secreted large amounts of capsular polysaccharide, increasing pathogenicity and hindering the separation of cells from the fermentation broth during downstream processing.     

Enhanced production of tetramethylpyrazine in <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> BL1 by <Emphasis Type="Italic">bdhA</Emphasis> disruption and 2,3-butanediol supplementation

The 2,3-butanediol (2,3-BD) dehydrogenase gene (bdhA) of Bacillus licheniformis BL1 was disrupted to construct the tetramethylpyrazine (TMP)-producing BLA strain. During microaerobic fermentation, the bdhA-disrupted BLA strain produced 46.98 g TMP/l, and this yield was 23.99 % higher than that produced by the parent BL1 strain. In addition, the yield of acetoin, which is a TMP precursor, also increased by 28.98 % in BLA. The TMP production by BL1 was enhanced by supplementing the fermentation medium with 2,3-BD. The yield of TMP improved from 37.89 to 44.77 g/l as the concentration of 2,3-BD increased from 0 to 2 g/l. The maximum TMP and acetoin yields increased by 18.16 and 17.87 %, respectively with the increase in 2,3-BD concentration from 0 to 2 g/l. However, no increase was observed when the concentration of 2,3-BD in the matrix was ≥3 g/l. This study provides a valuable strategy to enhance TMP and acetoin productivity of mutagenic strains by gene manipulation and optimizing fermentation conditions.     

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.     

2-Ketogluconic acid production by Klebsiella pneumoniae CGMCC 1.6366

Klebsiella pneumoniae CGMCC 1.6366 is a bacterium isolated for 1,3-propanediol or 2,3-butanediol production previously. K. pneumoniae ΔbudA, a 2,3-butanediol synthesis pathway truncated mutant with the gene deletion of budA which encodes alpha-acetolactate decarboxylase, was found to execrate an unknown chemical at a high titer when grown in the broth using glucose as carbon source. Later this chemical was identified to be 2-ketogluconic acid, which was formed through the glucose oxidation pathway in K. pneumoniae. It was found that 2-ketogluconic can also be produced by the wild strain. The fermentation studies showed that the production of this metabolite is strictly pH dependent, when the fermenting broth was maintained at pH 6–7, the main metabolite produced by K. pneumoniae CGMCC 1.6366 was 2,3-butanediol, or some organic acids in the budA mutated strain. However, if the cells were fermented at pH 4.7, 2-ketogluconic acid was formed, and the secretion of all other organic acids or 2,3-butanediol were limited. In the 5L bioreactors, a final level of 38.2 and 30.2 g/L 2-ketogluconic acid were accumulated by the wild type and the budA mutant K. pneumoniae, respectively, in 26 and 56 h; and the conversion ratios of glucose to 2-ketogluconic acid reached 0.86 and 0.91 mol/mol for the wild and the budA mutant, respectively.